Lecture 4 / 5 Flashcards
Nucleocytoplasmic transport
- some proteins are imported into nucleus without a ___. What is this referred to as?
- some proteins are imported into nucleus without an NLS
- referred to as ‘piggyback’ nuclear protein import * newly-synthesized protein lacking an NLS binds
to NLS-containing protein in cytoplasm
many proteins ‘shuttle’ between nucleus and cytoplasm - participate in both nuclear & cytoplasmic functions
* often contain both __
NLS and NES - relative distribution of protein in either compartment controlled by the relative strength of NLS and NES
strength of NLS or NES can be controlled by ___
post-translational modifications
Nucleocytoplasmic transport of ARC1
- ARC1 shuttles between ___ and ___
ARC1 shuttles between cytoplasm and nucleus
ARC1 possesses both an NLS and NES
- before pollination ?
- during (self-)pollination ?
- before pollination – (NLS > NES) results in ARC1 being
localized mostly in nucleus - during (self-)pollination - NLS disrupted due to phosphorylation of adjacent
amino acid residue(s)
(NLS Phos < NES) results in ARC1 localized mostly in cytoplasm
ARC1 in cytoplasm functions in proteosome-dependent turnover of other proteins
proteosome =
protein
degradation machinery [see later]
Nucleocytoplasmic transport of ARC1
What are the two experiments?
Experiment 1: Phosphorylation of NLS results in ARC1 (mis)localized to only cytoplasm
Introduce phosphomimic mutations to residues adjacent to NLS - aspartic acid [D] physicochemically similar to phosphorylated S/T/Y
Experiment 2: Mutation of NES results in ARC1 (mis)localized to only nucleus
In vitro co-immunoprecipitation assay to assess ‘cargo’ protein-importin binding
Co-immunoprecipitation (Co-IP) assay – two main components:
‘Bait’ – purified epitope-tagged nuclear protein (e.g., Myc-ARC1 or ARC1 missing its NLS [Myc-ARC1 NLS mut])
‘Prey’ – purified importin (a/b subunits)
Step 1: mix ‘bait’ and/or ’prey’ proteins in vitro
Step 2: add agarose beads coated with anti-epitope-tag IgGs
Step 3: isolate beads (via centrifugation) along with all associated (i.e., interacting) proteins
Step 4: SDS-PAGE and Coomassie blue staining
Two main phases in a cell cycle
- Interphase – three stages:
G1 - (Gap 1) cell performs normal cellular activities and can respond to environment
S - (Synthesis) DNA replication & increased synthesis of factors required for chromosome duplication (e.g., histones)
G2 - (Gap 2) cell grows & prepares for mitosis
* also… G0 (Gap 0) – non-dividing cells (most cells) - M phase – mitosis
consists of prophase, metaphase, anaphase and telophase - duplicated chromosomes separated into
two nuclei
consists also of cytokinesis - mother cell divides into two daughter cells
What is a checkpoint?
progression (or arrest) through cell cycle regulated at distinct stages
involve surveillance mechanisms that ensure cell cycle proceeds properly
several primary checkpoints in cell cycle:
*mid G1 (‘Restriction point’ or ‘START’) – cell commits to DNA replication in S and organelle duplication begins
*end of G2 – cell commits to entering M phase
*end of metaphase – cell commits to chromosome segregation
What happens if there is no checkpoint ?
cellular signals lead to cell death (apoptosis), cell cycle arrest (senescence), or disease (cancer)
Opened versus closed mitosis
‘Open’ Mitosis (higher eukaryotes)
Nucleus completely disassembles by metaphase and reassembles by telophase - all in coordination with DNA condensation/segregation during mitosis
‘Closed’ Mitosis’ (lower eukaryotes) Nucleus remains intact during mitosis
Main components of Standard brightfield microscope
light source, condenser lens, stage (holding specimen), objective and ocular (projection) lenses, and ‘detector’ (eye)
Image usually captured by a video camera, more sensitive to low light intensities
Easily manipulates data imaging, using various computer software example de convocation
Primary purpose of microscopy
to generate magnified, high-quality view of specimen
overall magnification = objective lens x ocular lens
Most important aspect of a microscope and it’s two main factors
Resolution is the most important aspect of today’s microscope
resolving power of microscope depends two main factors:
1. wavelength (l) of illumination light
2. numerical aperture (NA) - light-gathering qualities of objective lens and specimen
mounting medium
Major limitation of brake microscopy
specimen’s poor contrast
- specimens usually ‘fixed’ (e.g., formaldehyde fixation – cross-links amino groups on adjacent proteins/nucleic acids), embedded (in plastic or wax) for support, then sectioned with
microtome, and stained with molecule-specific dye(s)
What is Fluorescence microscopy used for?
- microscopy technique for visualizing fluorescent molecules in living (or fixed) specimens
- relies on endogenous fluorescence in specimen (autofluorescence), applied fluorescent dyes or dye-conjugated antibodies (immunofluorescence), and/or autofluorescent proteins
Principles of fluorescence
- certain atoms ( GFP in florescence molecule ) can absorb photon of certain l (e.g. blue light)
- atom’s electron becomes ‘excited’ and moves up to higher energy state
- ‘excited’ electron highly unstable
loses energy and returns to “ground state” by emitting
photon with lower energy (i.e., longer l) (e.g. red light) - ‘emitting’ electron has lower energy (longer wavelength) because some energy initially lost as heat
Confocal laser scanning microscopy
lasers can penetrate further into thicker living specimens
Z section vs Z stack
individual ‘z-sections’ collected at different depths in sample and combined
(i.e., z-sections stacked together serially) to form z-stack and generate 3D image
What are some limit confocal laser scanning microscopy?
rapid, but cannot capture very dynamic cellular processes
point laser light can photobleach fluorescent molecules (i.e., no longer fluorescent) and damage live cells by phototoxicity (i.e., ‘excited’ fluorescent molecules react with molecular oxygen to produce free radicals that damage membranes leading to cell death)
not efficient for imaging deep into thicker specimens/tissues
limited spatial resolution (~200 nm)
